THE PROTEASES

3 big problems for the kinetics:

H2O is a bad nucleophile
H O
NH is a bad leaving group
H loss of resonance during reaction

No problem for thermodynamics ( G << 0 )

N
N

H H O G < 0

H2O: H
O H
N - +
O H3 N

H O
 THE PROTEASES

H O H
N
N

H H O

H2O

The peptide bond is stable under physiological conditions

t1/2 102 years despite its thermodynamic instability

Hydrolyzed under very harsh conditions,

in acid (HCl 6 M, 110C, 24-72 h)
or base (KOH 1 M 100C 24 h)
 THE PROTEASES
3 big problems for the kinetics: H O H
H2O is a bad nucleophile
N
N
NH is a bad leaving group
loss of mesomery during reaction H H O
5 biological strategies to solve the problems, 5 classes of proteases
1. Serine proteases HO 2
Trypsin in mammalian digestion
Coagulation factors (Thrombine)

2. Cysteine proteases
Papaine
Cathepsines (in the lysosomes)

3. Aspartyl proteases
Pepsine (in our stomach)
AIDS virus protease

4. Metal ion proteases (Zn2+ )

Carboxypeptidases
5. Threonine proteases
Proteasome
 THE PROTEASES

Where the proteases act?

Exo-proteases (exo-peptidases, cut amino acids from
the N- or from the C-terminal of proteins/peptides)
Endo-proteases (cut in the interior of proteins/peptides)

Specificity
Non-specific (Proteinase K, used for stability studies of proteins)

Specific proteases (Trypsine X-X-ArgX-X et X-X-LysX-X

recognize 1 side chain
Very specific (Thrombine LeuValProArgGlySer )
recognize 6 side chains
 THE PROTEASES
IMPORTANT HINT! The protease best substrates are UNFOLDED proteins.
Compact protein domain are not hydrolyzed, instead the connecting domains
are hydrolyzed Essential application in protein biochemistry and imunology
for domain preparation by controlled proteolysis
Classical experiment: Porter 1955, preparation of immunoglobulin fragments
by treatment with papain or pepsin
Disulfide bonds
THE PROTEASES

SDS gel electrophoresis

 Stanley B. Prusiner
strange pathogen (resistance to UV, heat etc)
transmission to mouse (incubation time 150-300 days)
1975-77: transmission to hamster (70 days)
The presence of the Prion protein is demonstrated by the
resistence to proteolysis
infected
proteinase K

C SC
P rP P rP
P r P sen P r P 2 7 -3 0
P rP res

1 232
Blood clotting

Blood clotting results from a cascade of reactions.

In a cascade, a signal initiates a series of steps, each of them

catalyzed by an enzyme. At each step the signal is amplified.

In blood clotting the activated form of one clotting factor

catalyzes activation of the next.

Very small amounts of the initial factors trigger the cascade,

=> rapid response to trauma (e.g., damage to a blood vessel).
 Blood clotting

Fibrin fiber
 Conversion of fibrinogen to fibrin causes clotting.

The final step of clotting is conversion of fibrinogen to fibrin by

thrombin, a protease.

Fibrinogen has 6 protein chains (2x A, B and ), folded into

globular units connected by rods. Thrombin cleaves 4
peptides from the A and B chains in the central globule,
resulting in fibrin monomer ()2.
Carboxyl ends of the - and chains interact with the newly
exposed N-terminal regions => polymerization (protofibrils).
Blood Coagulation

Hemostasis
Fibrils are stabilized by cross-linking: formation of amide
bonds between lysine and glutamine by transglutaminase,
which is activated from protransglutaminase by thrombin.

The network of fibrils forms the clot.

 Activation of thrombin.

Thrombin activates fibrinogen, but how is thrombin activated ?

Thrombin is activated by proteolytic activation of prothrombin

with factors Xa (also a protease) and Va. Activation removes a
gla and 2 kringle domains.

Modular structure of prothrombin

 Use of chromogenic substrates for studying the proteases
Thrombine (enzyme in blood coagulation)

Natural substrate: le fibrinogen (a large protein, about 2000 residues)

Benzoyl-Phe-Val-Arg NH NO2

The product (p-nitro-aniline) est yellow

( 380 nm) H2N NO2
 1. The serine proteases

Proteases having an essential serine in the active site

H O H
Protases
Trypsine N
Chymotrypsine N
N

Elastase O
H H O
Subtilisine (Bacilus subtilis)
R
Same mechanism for esterases
Lipases H2O
H O
Esterases H
(actyl)choliesterase
N
O
Amides and esters have similar H O
structure and reactivity
H2O
 Identification of active serine in serine proteases

An Unusually Reactive Serine in Chymotrypsin

Chymotrypsin is inactivated by treatment with
diisopropylphosphofluoridate (DIPF), which reacts only with serine
195 among 28 possible serine residues. No reaction with the
unfolded enzyme, nor with free serine
 Identification of active serine in serine proteases

Addition of Substrate protects DIFP Inhibition

100 No substrate
+ DIFP
Percent Inhibition of activity (%)

50

+ DIFP & substrate Add substrate

S
0
Reaction time
 Evidence for Histidine Participation
O O
CH2 CH C OCH3 CH2 CH C CH2Cl
NH NH
SO2 SO2

CH3 CH3
2.11 2.12

substrate inactivator
(TPCK)
With [14C]TPCK get 1 equiv. [14C] bound; pepsin hydrolysis
gives a [14C] peptide with His-57 modified
The serine proteases: the specificity pocket
4 Catalytic elements in serine proteases

O-
4 Catalytic elements in serine proteases

Specificity pocket
Aa 189, 216, 226

Oxyanion hole
Aa 193-195

Substrate binding
Aa 214-216

Catalytic triad
Ser195, His57, Asp102

Chymotrypsin
Chymotrypsin
STRUCTURE: David BLOW 1968
:

Serine is the NUCLEOPHILE

Histidine is a BASE: it binds the serines
proton and decreases its pKa from 15 to
about 7
The aspartate keeps the histidine in the
correct orientation (an old theory: proton
relay, but the proton does not move)
 but identical active site!

Subtilisin

An example of CONVERGENT evolution

Trypsin
volution convergente
CONVERGENT evolution
Thrombin and Chymotrypsin are
HOMOLOGS
(Almost identical structures, similar sequences
 Evolution is most often DIVERGENT

Ancestral gene, duplication

and separate evolution by mutation

Trypsine, chymotrypsine, lastase

Structure trs similaire
Famille de protines
Triade: Ser195, His57, Asp102

Different genes, protein evolution

To a similar active site configuration

A few examples of Subtilisine

Structure trs diffrente
CONVERGENT evolution Triade: Ser221, His64, Asp32
Many serine proteases age activated by proteolysis
(protection of the cells which synthetize the proteases)
 Serine Protease Mechanism - Chymotrypsin

Hydrophobic
Active pocket
site
residues
Disulfide bridges
This is a reaction
INTERMEDIATE Reaction coordinate
and not a transition
state
The C-terminal part of the
substrate dissociated and
leaves the Acyl-enzyme
STEP 2: Acyl-enzyme
hydrolysis
Kinetic demonstration of the serine protease mechanism: burst kinetics
 Demonstration of the serine protease mechanism: site-directed mutagenesis

Nature. 1988, 332(6164):564-8.

Substrate: N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide

Bacillus amyloliquefaciens subtilisin, these functional elements

impart a total rate enhancement of at least 109 to 1010 times the
non-enzymatic hydrolysis of amide bonds
 Reaction mechanism of a serine protease
(in this case, subtilisin)

Note the three residues of the catalytic triad: Ser221, His64, & Asp32.

 Demonstration of the serine protease mechanism: site-directed mutagenesis

Subtilisine Km kcat/Km kcat

Asp32 H64 S221 (M) (M-1 s-1) s-1
Asp His Ser 220 250000 55
Ala His Ser 480 5 0.0024
Asp Ala Ser 390 0.1 0.000039
Asp His Ala 420 0.1 0.000042
Ala Ala Ala 420 0.1 0.000042

kSerHisAsp/knon-enzymatique = 3 750 000 000

kAlaAlaAla/knon-enzymatique = 3 000
1. When very low residual activities are expected, a very low level
of contamination with other proteases is a serieus problem.
How has this been avoided? Serine24 (on the protein surface)
has been replaced by a Cysteine which makes possible protein
purification by covalent affinity chromatography.

2. A second problem could be the mis-incorporation during

traduction. An error rate of 1/1000 can be a problem !
3. Ascertaining the role of specific amino acids in catalysis by
site-directed mutagenesis can easily by interpreted if the
chemical step is rate-limiting (A). If the substrate binding is rate-
limiting (B), it is well possible to miss important details of the
mechanism. The measured rate is slower with the mutant
No apparent effect!
A B
G G

G G
E+S E+S

G G
ES ES
E+P E+P

Reaction coordinate Reaction coordinate

Replacing an active-site residue will slown down reaction in A but
not in B
Take home lesson: even with no catalytic residues, the enzyme still
accelerates the reaction better than 1000-fold the rate of the
uncatalyzed reaction. Way to bind that transition state!
 Demonstration of the serine protease mechanism: site-directed mutagenesis
Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.
Bryan P, Pantoliano MW, Quill SG, Hsiao HY, Poulos T.
Proc Natl Acad Sci U S A. 1986 Jun;83(11):3743-5.

Reaction intermediate is Reaction intermediate is

stabilized by main-chain NH stabilized Asn side-chain in
in chymotrypsin: its role subtilisin: its role CAN be
cannot be probed by site- probed by site-directed
directed mutagenesis mutagenesis!!!
 Demonstration of the serine protease mechanism: site-directed mutagenesis
In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed
is believed to adopt a tetrahedral configuration rather than the ground-state planar
configuration. Crystallographic studies suggest that stabilization of this activated
complex is accomplished in part through the donation of a hydrogen bond from the
amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To
specifically test this hypothesis, leucine was introduced at position 155. Leucine is
isosteric with asparagine but is incapable of donating a hydrogen bond to the
tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a
greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed
with a peptide substrate. These kinetic results are consistent with the Asn-155
mediating stabilization of the activated complex and lend further experimental support
for the transition-state stabilization hypothesis of enzyme catalysis.
A recent addition to the serine protease mechanism:
the Low barrier hydrogen bonds

2.8 2.55 2.29

 A recent addition to the serine protease mechanism: the Low
barrier hydrogen bonds
Low-Barrier Hydrogen Bonds and Enzymic Catalysis
W. W. Cleland and Maurice M. Kreevoy

Formation of a short (less than 2.5 angstroms), very strong, low-

barrier hydrogen bond in the transition state, or in an enzyme-
intermediate complex, can be an important contribution to enzymic
catalysis. Formation of such a bond can supply 10 to 20 kilocalories
per mole and thus facilitate difficult reactions such as enolization of
carboxylate groups. Because low-barrier hydrogen bonds form only
when the pKa's (negative logarithm of the acid constant) of the
oxygens or nitrogens sharing the hydrogen are similar, a weak
hydrogen bond in the enzyme-substrate complex in which the
pKas do not match can become a strong, low-barrier one if the
pKas become matched in the transition state or enzyme-
intermediate complex.
A second recent addition to the serine protease mechanism:
Substrate assisted catalysis
A recent addition to the serine protease mechanism
Substrate assisted catalysis
A second recent addition to the serine protease mechanism
Substrate assisted catalysis
A recent addition to the serine protease mechanism
Substrate assisted catalysis
Can proteases be used for protein SYNTHESIS?

CHEMICAL ligation
Kaiser and co-workers demonstrated the practicality of this work
by preparing a subtilisin variant, thiolsubtilisin, where the active
site Ser was chemically converted to Cys (S221C)) Using
activated esters to acylate the active site Cys in the presence of
amine nucleophiles, it was possible to efficiently synthesize
amide bonds. The ratio of aminolysis to hydrolysis is 600-fold
greater for thiolsubtilisin relative to subtilisin; the variant
selenolsubtilisin was later prepared by Hilvert and co-workers and
shown to be 14,000-fold more effective for aminolysis than
subtilisin.
 Hydrolysis

Aminolysis

Aminolysis/Hydrolysis

Serine OH 1.0
Cysteine SH 600
Selenocysteine SeH 14000 Meth Enz 289, 298-313
Subtiligase: a tool for semisynthesis of proteins.
Chang TK, Jackson DY, Burnier JP, Wells JA.
Department of Protein Engineering, Genentech, Inc., South San Franc
94080.
 Serine hydrolases: proteases and other enzymes

Serine proteases

Asparaginase

Esterase

Penicillin acylase

-lactamase
The acetyl-cholinesterase a serine esterase
Acetylcholinesterase: an archetype for cationp bonding in biology?

Acetylcholinesterase is often considered as the foremost example of

cationp bonding in biological molecular recognition. In its interaction
with acetylcholine, it serves as an excellent model for the recognition of
quaternary amines by proteins. Early kinetic, spectroscopic and
chemical modification studies [17] suggested that the active site of
acetylcholinesterase is divided into two subsites: the 'esteratic' site (the
site of bond breaking/making) and the 'anionic' (choline binding) site.
The 'anionic' site is a misnomer, as this site is in fact uncharged and
lipophilic. The molecular detail of acetylcholinesterase was revealed
following the determination of the crystal structure of the enzyme from
Torpedo californicans [18]. A structure for the enzymesubstrate
complex is not available, but the details of substrate binding can be
extrapolated from the structure of the enzyme alone [18] and those of
the enzyme complexed with tacrine, edrophonium and decamethonium
[19].
 2. Cysteine proteases

Papane from plants is one example

Cathepsines (protease from lysosomes)

S
H :N N H
 3. Aspartyl proteases

Protease from AIDS virus: an aspartyl protaase

 3. Aspartyl protases

Isovaleryl-Val- Val- Sta- Ala- Sta

statine
Pepstatin is a potent inhibitor of aspartyl proteases. It is a hexa-
peptide containing the unusual amino acid statine (Sta, (3S,4S)-4-
amino-3-hydroxy-6-methylheptanoic acid), having the sequence
Isovaleryl-Val-Val-Sta-Ala-Sta (Iva-Val-Val-Sta-Ala-Sta). It was
originally isolated from cultures of various species of Actinomyces
due to its ability to inhibit pepsin at picomolar concentrations. It
was later found to inhibit nearly all acid proteases with high
potency and, as such, has become a valuable research tool, as
well as a common constituent of protease inhibitor cocktails.
This is a TRANSITION STATE ANALOG
 4. Metallo-proteases

carboxypeptidase A
5. A new mechanism: the threonine protease in the proteasome
During proteasome-catalysed transpeptidation, the energy from peptide-bond hydrolysis fuels subsequent
peptide-bond ligation. When presented with the three- and six-residue components of the nine-residue peptide,
the proteasome was unable to splice them together. However, when supplied with the six and seven-residue
fragments that comprise the 13-residue precursor peptide, the proteasome efficiently produced the nona-peptide.
These observations indicated that the proteasome can catalyse peptide-bond
formation only when the process is linked to peptide-bond
hydrolysis. Nucleophilic attack of peptide bonds by the hydroxyl
group of an active-site threonine in the proteasome results in an
acyl-enzyme intermediate, in which the peptide and the threonine
are joined by an ester bond. The acyl-enzyme intermediate plays
a part in the proteasome-catalysed transpeptidation event. In the
first step the hydroxyl group of an active-site threonine catalyses
the cleavage of a precursor peptide, generating an N-terminal and
a C-terminal fragment. In the second step an active-site threonine
attacks the peptide bond in the N-terminal fragment forming an
acyl-enzyme
The architecture ofintermediate
the central chamber with the N-terminal
of the proteasome peptide.
defines the catalytic Atandthis
specificity point
also might
the N-terminus
regulate of the
the incidence of splicing. TheC-terminal
substrate-binding peptide fragment
sites that flank the scissile bondattacks the
favour certain amino
acids and, therefore, enable certain peptides to linger in the active-site cavity, thus providing an opportunity for
acyl-enzyme
an N-terminal nucleophileintermediate and, intermediate.
to attack the acyl-enzyme recycling Thethe energy
determinants from the
for protease-catalysed
cleavage
splicing reaction,
are certainly ligates
finely controlled becauseonto thesite(now
the active also mustcleaved)
enable normalN-terminal
proteolytic events to occur.
The question that arises in the case of proteasome-catalysed protein splicing is whether the splicing process is
peptide
favoured for .aThis transpeptidation
functional purpose ofmodel explains
the resulting how peptide-bond
peptides. Proteasomes hydrolysis
may notandonlyformation
mediate occur together
the complete
without the net
degradation input of energy.
of proteins, but alsoIt the
shows also thatofthe
processing splice site
precursors need
into not be
mature, highly
active conserved because, once a
proteins.
peptide bond has been activated at the protease active site, ligation of almost any incoming peptide with a free N-
 Protein Splicing: Analogy to RNA Splicing

Attention: this is different from typical enzyme in that it is

single turn-over!
 Properties of protein splicing

1. Protein splicing is catalyzed entirely by

amino acid residues contained in the
intein.

2. Protein splicing is an intramolecular

process (usually).

3. Protein splicing requires no coenzymes or

sources of metabolic energy and therefore
involves bond rearrangements rather than
bond cleavage followed by resynthesis.

Annu Rev Biochem. 2000;69:447-96. Protein splicing and related forms of protein autoprocessing. Paulus H.
 What do they look like?

Small inteins are about 150 amino acids.

(the smallest is 134 amino acids, largest is 1650)
Step 1: formation of a linear ester intermediate by NO or
NS acyl rearrangement involving the nucleophilic amino
acid residue at the N-terminal splice junction;

Step 2: formation of a branched ester intermediate by

the attack of the nucleophilic residue at the C-terminal
splice junction on the linear ester intermediate;

Step 3: cyclization of the asparagine residue adjacent to

the C-terminal splice junction, coupled to cleavage of
the branched ester intermediate to yield an excised
intein with a C-terminal aminosuccinimide residue and
the two exteins joined by an ester bond;

Step 4: spontaneous hydrolysis of the aminosuccinimide

residue and rearrangement of the ester linking the
exteins to the more stable amide bond.

The last step is spontaneous and irreversible.

The first three steps are catalyzed by the intein
Annu Rev Biochem. 2000;69:447-96.
Protein splicing and related forms of protein autoprocessing.
Paulus H.
Same chemistry as protein splicing has been used for
spontaneous (non-enzymatic) peptide ligation
Dawson PE, Muir TW, Clark-Lewis I, Kent SB.
Synthesis of proteins by native chemical ligation.
Science. 1994 Nov 4;266(5186):776-9.
Proposed mechanism of amide, true peptide, and ester bond hydrolysis by proteasomes and
mechanism of their inactivation by irreversible inhibitors.

Kisselev A F et al. J. Biol. Chem. 2000;275:14831-14837

INHIBITORS
 3. Aspartyl protases
Une des composantes de la tri-thrapie est un inhibiteur de la
Protase du virus du SIDA, un analogue de ltat de transition
Access to the active site of acetylcholinesterase is via a deep and narrow gorg
up about 40% of the surface of the gorge) and other residues. The gorge is 20
the surface of the gorge are highly conserved in acetylcholinesterases from di
substrate acetylcholine at the base of the gorge reveals the esteratic and chol
esteratic site, a catalytic triad and putative oxyanion hole have been identified
acetylcholine suggests that it forms a cationp bond with Trp-84 in the 'anionic
remarkable feature of acetylcholinesterase is the preponderance of aromatic r
chemical character of the gorge leads to the question of its function in contribu
and catalysis. Sussman and colleagues suggested two mechanisms by which
increased [18]. First, the high hydrophobicity of the gorge produces a low diele
to enhance the effective local charge contributed by the small number of acidi
electrostatically 'steer' substrate to the active site. In the second scenario, the
affinity sites for the substrate (in particular, the choline moiety), and guides the
Because of the reduction-in-dimensionality, the rate of substrate binding is inc
interactions may, therefore, have a major role to play in directing the substrate
substrate complex, whereas stronger cationp bonding is presumably respons
acetylcholine in the enzymesubstrate complex. Given the wealth of cationp
the enzyme no doubt will remain a principal target for investigating these inter
Interestingly, chemical modification studies of the nicotinic acetylcholine recep
residues are located in the acetylcholine-binding site in this molecule [20,21].
Leupeptin, also known as N-acetyl-L-leucyl-L-leucyl-L-
argininal, is a protease inhibitor that also acts as an inhibitor
of calpain.

It is often used during in vitro experiments when a specific

enzymatic reaction is being studied. When cells are lysed
for these studies, proteases, many of which are contained
within lysosomes, are released. These proteases, if freely
present in the lysate, would destroy any products from the
reaction being studied, and make the experiment
uninterpretable. For example, leupeptin could be used in a
calpain extraction to keep calpain from being hydrolyzed by
specific proteases. The suggested concentration is 1-10 M
(0.5-1 g/ml).
Leupeptin is an organic compound produced by
 3. Aspartyl protases
Une des composantes de la tri-thrapie est un inhibiteur de la
Protase du virus du SIDA, un analogue de ltat de transition